S. K. Chauthe et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2251–2256
2255
(CHCl3): kmax (log
e
) 285 (4.49), 340 (4.15); IR (Neat): mmax 3403, 2949, 1615,
1H NMR (CDCl3, 300 MHz): d
References and notes
1581, 1459, 1399, 1459, 1399, 1195, 1044 cmÀ1
;
17.94 (br s, 2H, 2Â OHC), 16.46 (s, 2H, 2Â OHB), 10.70 (br s, 1H, OHA), 10.22 (br s,
1H, OHA), 4.80 (q, J = 7.2 Hz, 1H), 3.05 (d, J = 6.5 Hz, 8H), 2.27 (m, 4H), 1.79 (d,
J = 7.2 Hz, 3H), 1.00 (d, J = 6.4 Hz, 24H); 13C NMR (CDCl3, 75 MHz): d 208.5, 208.0,
171.2, 167.2, 163.1, 109.2, 105.7, 104.7, 53.9, 52.9, 25.7, 23.3, 17.7; CIMS: m/z
321[MÀC10H10O5]; analysis for C34H46O10 (614.3), calcd, C, 66.43; H, 7.54; found,
C, 66.52; H, 7.49. Ethyl-methylene-bis-(3,5-di-isopentanoyl-2,4,6-trihydroxybenzene)
1. WHO’s fight against cancer: strategies that prevent, cure and care 2007, http://
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(14): Yield: 41%; yellow oil; UV (CHCl3): kmax (log
e
) 285 (4.48), 337 (4.13); IR (Neat):
mmax 3199, 2960, 2873, 1616, 1584, 1466, 1367, 1367, 1301, 1200, 1047 cmÀ1
;
1H
NMR (CDCl3, 300 MHz): d 18.00 (s, 1H, OHC), 17.76 (s, 1H, OHC), 16.45 (s, 2H, 2Â OHB),
10.87 (s, 1H, OHA), 10.06 (s, 1H, OHA), 4.44 (t, J = 7.7 Hz, 1H), 3.06 (d, J = 6.5 Hz, 8H),
2.27 (m, 6H), 1.00 (d, J = 6.4 Hz, 24H), 0.87 (t, J = 7.2 Hz, 3H, overlapped); 13C NMR
(CDCl3, 75 MHz): d 208.7, 208.1, 171.2, 169.6, 166.1, 107.9, 105.7, 104.6, 53.9, 52.9,
32.7, 25.7, 24.3, 23.3, 12.8; CIMS: m/z 335[MÀC10H10O5]; analysis for C35H48O10
(628.3), calcd, C, 66.86; H, 7.69; found, C, 66.70; H, 7.82. Cyclohexyl-methylene-bis-
(3,5-di-isopentanoyl-2,4,6-trihydroxybenzene) (15): Yield: 33%; reddish brown solid;
mp 158–160 °C; UV (CHCl3): kmax (log
e
) 282 (4.49), 341 (3.90); IR (KBr):
mmax 3199,
2957, 2930, 1618, 1579, 1467, 1365, 1300, 1197, 1052 cmÀ1
;
1H NMR (CDCl3,
300 MHz): d 18.04 (s, 1H, OHC), 17.74 (s, 1H, OHC), 16.46 (s, 1H, OHB), 16.42 (s, 1H,
OHB), 10.96 (s, 1H, OHA), 10.07 (s, 1H, OHA), 4.17 (d, J = 10.8 Hz, 1H), 3.01 (br s, 8H),
2.82 (m, 1H), 2.27 (m, 4H), 1.74–1.52 (m, 6H), 1.00 (d, J = 5.7 Hz, 24H), 0.80–0.74 (m,
4H); 13CNMR(CDCl3,75 MHz):d208.2, 208.0, 171.1, 169.1, 163.3, 166.4, 166.0, 107.4,
107.1, 105.6, 104.5, 53.9, 53.0, 43.8, 37.5, 36.6, 32.9, 30.3, 26.8, 26.6, 25.9, 25.7, 23.3;
CIMS: m/z 683 [M+1]+, 389 [MÀC10H10O5]; analysis for C39H54O10 (682.4), calcd, C,
68.60; H, 7.97; found, C, 68.49; H, 8.06. Phenyl-methylene-bis-(3,5-diacetyl-2,4,6-
trihydroxybenzene) (16): Yield: 30%; cream colored solid; mp 109–111 °C; UV
(CHCl3): kmax (log
e
) 272 (4.22), 339 (3.39); IR (KBr): mmax 2925, 2676, 2562, 1688,
1603, 1584, 1455, 1424, 1327, 1292, 1180, 1128, 1073, 1027 cmÀ1
;
1H NMR (CDCl3,
300 MHz): d 17.65 (s, 2H, 2Â OHC), 16.37 (s, 2H, 2Â OHB), 10.21 (s, 2H, 2Â OHA), 7.30–
7.23 (m, 3H), 7.10 (d, J = 7.3 Hz, 2H), 6.17 (s, 1H), 2.78 (s, 6H), 2.72 (s, 6H); 13C NMR
(CDCl3, 75 MHz): d 205.9, 205.6, 171.6, 169.2, 166.6, 137.5, 128.9, 127.1, 127.0, 106.5,
105.8, 104.9, 34.0, 33.7, 32.9, 30.2; CIMS: m/z 299 [MÀC10H10O5]; analysis for
C27H24O10 (508.1), calcd, C, 63.78; H, 4.76; found, C, 63.64; H, 4.65. Thiophen-
2-yl-methylene-bis-(3,5-diacetyl-2,4,6-trihydroxybenzene) (17): Yield: 42%; yellowish
) 272 (4.29), 334 (3.65); IR (KBr):
General method for synthesis of diacyl and dimeric phloroglucinols using
conventional heating: The mixture of phloroglucinol (1, 1 mmol), acid
anhydride/acyl chloride (3 mmol), acetic acid (1 mmol) and methanesulfonic
acid (3 mmol) was heated for 30 min at 80 °C. On cooling, water was added and
the reaction mixture and was extracted with ethyl acetate. Crude diacyl
phloroglucinol derivative were crystallised from methanol. In case of synthesis
of dimeric phloroglucinol, after 30 min, the linker aldehyde was added to the
reaction mixture and the reaction was continued for further 15 min. After
completion of reaction (TLC), water was added to the reaction mixture and
extracted with ethyl acetate. Organic layer was dried over sodium sulphate and
the solvent was removed under vacuum and the crude product was purified by
crystallisation from methanol. General method for synthesis of diacyl
phloroglucinol derivative using microwave (IFB, model: 30SC2 with functions to
control heating) heating: The mixture of phloroglucinol (1, 1 mmol), acid
anhydride/acyl chloride (3 mmol), acetic acid (1 mmol) and methanesulfonic
acid (30 mol %) was irradiated in microwave oven for 1 min. The reaction
workup is similar to the conventional heating method. The yields are reported
less than 50% as two equivalents of monomeric starting material produce
1 equiv of dimeric product. Cell proliferation assay (CCK-8 assay): All the cancer
cells, Panc1, ACHN, H460, Calu1, HCT116, and MCF10A were obtained from the
American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained
in Dulbecco’s Modified Eagle Medium (DMEM, Sigma St. Louis, MO),
supplemented with 10% fetal bovine serum Gibco (Paisley, Scotland), 100 U/
ml penicillin (Sigma, St. Louis, MO) and 100-mg/ml streptomycin (Sigma St.
Louis, MO). The cells were grown in 75-cm2 culture flasks kept in a humidified
(37 °C, 5% CO2) incubator, and passaged on reaching 80% confluence. The assay
is based on the reduction of water-soluble tetrazolium salt (WST-8; [2-(2-
methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)]-2H-
tetrazolium mono sodium salt) by dehydrogenase enzyme of live cells to give a
yellow coloured product (formazan). 5 Â 103 cells were seeded per well in a 96
well tissue culture grade plate and the plate was kept overnight at 37 °C in 5%
CO2 incubator. After overnight incubation, cells were treated with different
concentrations of compounds/standard. An equal concentration of vehicle
(DMSO-never exceeding 0.1%) was used as a control. At 48 h after treatment,
green solid; mp 178–180 °C; UV (CHCl3): kmax (log
e
mmax 3179, 1616, 1579, 1481, 1409, 1365, 1262, 1186, 1116; 1H NMR (CDCl3,
300 MHz): d 17.80 (s, 2H, 2Â OHC), 16.39 (s, 2H, 2Â OHB), 10.32 (s, 2H, 2Â OHA), 7.19
(d, J = 5.0 Hz, 1H), 6.91 (t, J = 4.0 Hz, 1H), 6.70 (s, 1H), 6.26 (s, 1H), 2.78 (s, 6H), 2.73 (s,
6H); 13CNMR(CDCl3,75 MHz):d206.0, 205.7, 171.8, 169.2, 166.3, 142.9, 127.0, 125.4,
124.9,106.9,106.8, 105.4, 34.0, 32.8, 31.1; CIMS: m/z 305 [MÀC10H10O5]; analysis
for C25H22O10S (514.1), calcd, C, 58.36; H, 4.31; S, 6.23; found, C, 58.22; H, 4.44; S,
6.11.Thiophen-2-yl-methylene-bis-(3,5-di-isopentanoyl-2,4,6-trihydroxybenzene)
(18): Yield: 51%; dark yellow solid; mp 170–172 °C; UV (CHCl3): kmax (loge) 286
(4.56), 340 (3.99); IR (KBr):
mmax 3172, 2958, 2870, 1615, 1577, 1470, 1434, 1202,
1169, 1128, 1067 cmÀ1
;
1H NMR (CDCl3, 300 MHz): d 18.06 (s, 2H, 2Â OHC), 16.60
(s, 2H, 2Â OHB), 10.33 (s, 2H, 2Â OHA), 7.19 (d, J = 5.1 Hz, 1H), 6.92 (dd, J = 3.6,
5.0 Hz, 1H), 6.70 (t, J = 1.6 Hz, 1H), 6.25 (s, 1H), 3.05 (d, J = 6.1 Hz, 8H), 2.27 (m,
4H), 1.00 (d, J = 4.9 Hz, 24 H); 13C NMR (CDCl3, 75 MHz): d 207.9, 207.6, 171.3,
168.9, 165.4, 142.8, 126.4, 124.9, 124.3, 106.6, 105.2, 104.3, 53.3, 52.3, 31.2, 25.2,
23.3; CIMS: m/z 390 [MÀC10H10O5]; analysis for C37H46O10S (682.3), calcd, C,
65.08; H, 6.79; S, 4.70; found, C, 65.19; H, 6.87; S, 4.59. Pyridin-2-yl-methylene-
bis-(3,5-diacetyl-2,4,6-trihydroxybenzene) (19): Yield: 44%; brown solid; mp
230–232 °C; UV (CHCl3): kmax (log
e
) 276 (4.32), 339 (3.53); IR (KBr):
mmax 3096,
2933, 1610, 1454, 1360, 1294, 1219, 1117, 1028 cmÀ1
;
1H NMR (DMSO-d6,
300 MHz): d 17.09 (s, 1H, OH), 8.82 (d, J = 5.3 Hz, 1H), 8.56 (br s, 1H), 8.00 (s, 1H),
7.84 (d, J = 7.9 Hz, 1H), 6.94 (s, 1H), 2.86 (s, 6H), 2.81 (s, 6H); 13C NMR (DMSO-d6,
75 MHz): d 204.1, 172.7, 160.1, 145.2, 143.1, 125.2, 124.0, 106.0, 33.4, 32.8;
CIMS: m/z 510 [M+1]+, 300 [MÀC10H10O5]; analysis for C26H23NO10 (509.1), calcd,
C, 61.30; H, 4.55; N, 2.75; found, C, 61.21; H, 4.39; N, 2.60. 2,4-bis-(triflouromethyl)-1-
yl-methylene-bis-(3,5-di-isopentanoyl-2,4,6-trihydroxybenzene) (20): Yield: 40%;
yellow crystals, mp 211–213 °C, 1H NMR (CDCl3, 300 MHz): d7.93 (s, 1H), 7.75 (d,
J = 8.1 Hz, 1H), 7.48 (d, J = 8.1 Hz, 1H), 6.59 (s, 1H), 2.99–3.07 (Overlapped signals,
8H), 2.26 (m, 4H) , 0.99 (d, J = 6.6 Hz, 24H) 13C NMR (CDCl3, 75 MHz): d 208.4, 208.1,
207.5, 207.2, 171.6, 168.5, 166.1, 165.6, 142.2, 130.2, 130.00, 129.7, 129.3, 129.08,
128.8, 128.4, 126.1, 125.7, 124.9, 122.4, 122.1, 106.7, 105.7, 104.9, 53.9, 53.5, 52.9,
30.1, 25.6, 23.2 ; MALDI-TOF: m/z 812.02. 4-Biphenyl-1-yl-methylene-bis-(3,5-di-
isopentanoyl-2,4,6-trihydroxybenzene) (21): Yield: 43%; yellow crystals, mp 227–
229 °C, 1H NMR (CDCl3, 300 MHz): d 17.95 (br s, 2H, 2Â OHC), 16.57 (s, 2H, 2Â OHB),
10.32 (br s, 2H, 2Â OHA), 7.57 (d, J = 7.3 Hz, 2H), 7.49 (d, J = 8.1 Hz, 2H), 7.40 (t, 2H),
7.30 (d, J = 7.2 Hz, 1H), 7.15 (d, J = 8.0 Hz, 2H), 6.18 (s, 1H), 3.03 (d, J = 12 Hz, 8H), 2.27
(m, 4H), 0.99 (d, J = 5.5 Hz, 24 H); 13C NMR (CDCl3, 75 MHz): d 208.4, 208.2, 171.6,
169.4, 166.2, 141.1, 139.8, 136.8, 129.3, 127.7, 127.6, 127.5, 106.7, 105.8, 104.9,
53.8, 52.9, 33.7, 25.7, 23.3; MALDI-TOF: m/z 751.47. calcd, C, 71.79; H, 6.96;
found, C, 71.75.; H, 6.94. Benzofuran-2-yl-methylene-bis-(3,5-di-isopentanoyl-2,4,6-
trihydroxybenzene) (22): Yield: 41%; yellow crystals, mp 186–188 °C, IR (KBr): mmax
3435, 2958, 1614, 1580, 1466, 1301, 1199, 1108, 1H NMR (CDCl3, 300 MHz): d 17.95
(br s, 2H, 2Â OHC), 16.57 (s, 2H, 2Â OHB), 10.32 (br s, 2H, 2Â OHA), 7.48 (d, 1H), 7.33 (d,
1H), 7.16–7.21 (m, 2H), 6.42 (s, 1H), 6.17 (s, 1H), 2.92 (d, J = 12 Hz, 8H), 2.28 (m, 4H),
0.98 (d, J = 5.5 Hz, 24 H); 13C NMR (CDCl3, 75 MHz): d 208.4, 208.2, 172.1, 169.2,
165.8, 155.4, 129.0, 125.5, 124.2, 123.6, 120.9, 111.4, 110.1, 105.7, 105.4, 53.8, 52.7,
37.3, 25.4, 23.3; MALDI-TOF: m/z 717.4. Flourene-2-yl-methylene-bis-(3,5-di-
isopentanoyl-2,4,6-trihydroxybenzene (23): Yield: 40%; yellow crystals, mp 204–
206 °C; 1H NMR (CDCl3, 300 MHz): d 17.95 (br s, 2H, 2Â OHC), 16.60 (s, 2H, 2Â OHB),
10.32 (br s, 2H, 2Â OHA), 7.72 (d, J = 7.2 Hz, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.49 (d, J = 7.2,
1H), 7.32–7.25 (m, 3H), 7.11 (d, J = 8.1, 1H), 6.24 (s, 1H), 3.81 (s, 2H), 3.06 (d,
aliquots of 5 ll of CCK-8 reagent were added to each well and incubated for 4 h
at 37 °C. After appropriate incubation, the absorbance was measured on Tecan
Sapphire multi-fluorescence micro-plate reader (Tecan, Germany, GmbH) at a
wavelength of 450 nm corrected to 650 nm and normalized to controls. DMSO
treated control cells were considered to have a cell viability of 100%. The
average number of viable cells at different compound/standard concentrations
was expressed as a percentage of the control.
Methyl-methylene-bis-(3,5-diacetyl-2,4,6-trihydroxybenzene) (12): Yield: 32%;
yellow solid; mp 206–208 °C; UV (CHCl3): kmax (loge) 276 (4.37), 341 (3.92);
IR (KBr): mmax 3168, 1618, 1579, 1476, 1419, 1364, 1263, 1185, 1115,
1024 cmÀ1
;
1H NMR (CDCl3, 300 MHz): d 17.65 (s, 2H, 2Â OHC), 16.25 (s, 2H,
2Â OHB), 10.71 (br s, 1H, OHA), 10.22 (br s, 1H, OHA), 4.80 (q, J = 7.2 Hz, 1H),
2.76 (s, 6H), 2.73 (s, 6H), 1.79 (d, J = 7.2 Hz, 3H); 13C NMR (CDCl3, 75 MHz): d
206.0, 205.7, 171.2, 168.2, 166.2, 108.9, 105.7, 104.6, 34.0, 32.9, 24.2, 17.6;
CIMS: m/z 447 [M+1]+, 237 [MÀC10H10O5]; analysis for C22H22O10 (446.1),
calcd, C, 59.19; H, 4.97; found, C, 59.10; H, 5.04. Methyl-methylene-bis-(3,5-di-
isopentanoyl-2,4,6-trihydroxybenzene) (13): Yield: 37%; yellow sticky mass; UV